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Journal of Clinical Microbiology, January 2007, p. 134-140, Vol. 45, No. 1
0095-1137/07/$08.00+0     doi:10.1128/JCM.01544-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Identification of Vibrio Isolates by a Multiplex PCR Assay and rpoB Sequence Determination

Cheryl L. Tarr,^1,2* Jayna S. Patel,¹ Nancy D. Puhr,¹ Evangeline G. Sowers,¹ Cheryl A. Bopp,¹ and Nancy A. Strockbine¹

Foodborne and Diarrheal Diseases Branch, Division of Bacterial and Mycotic Diseases, National Center for Infectious Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia 30333,¹ Battelle Memorial Institute, Battelle Science and Technology International, Chemical and Environmental Technologies, Atlanta Analytical Chemistry Group, 2971 Flowers Road South, Atlanta, Georgia 30341²

Received 25 July 2006/ Returned for modification 1 September 2006/ Accepted 2 November 2006

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

 
Vibrio, a diverse genus of aquatic bacteria, currently includes 72 species, 12 of which occur in human clinical samples. Of these 12, three species—Vibrio cholerae, Vibrio parahaemolyticus, and Vibrio vulnificus—account for the majority of Vibrio infections in humans. Rapid and accurate identification of Vibrio species has been problematic because phenotypic characteristics are variable within species and biochemical identification requires 2 or more days to complete. To facilitate the identification of human-pathogenic species, we developed a multiplex PCR that uses species-specific primers to amplify gene regions in four species (V. cholerae, V. parahaemolyticus, V. vulnificus, and V. mimicus). The assay was tested on a sample of 309 Vibrio isolates representing 26 named species (including 12 human pathogens) that had been characterized by biochemical methods. A total of 190 isolates that had been identified as one of the four target species all yielded results consistent with the previous classification. The assay identified an additional four V. parahaemolyticus isolates among the other 119 isolates. Sequence analysis based on rpoB was used to validate the multiplex results for these four isolates, and all clustered with other V. parahaemolyticus sequences. The rpoB sequences for 12 of 15 previously unidentified isolates clustered with other Vibrio species in a phylogenetic analysis, and three isolates appeared to represent unnamed Vibrio species. The PCR assay provides a simple, rapid, and reliable tool for identification of the major Vibrio pathogens in clinical samples, and rpoB sequencing provides an additional identification tool for other species in the genus Vibrio.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

 
The genus Vibrio is a highly diverse group of gram-negative bacteria that contains approximately 72 species (www.bacterio.net). The group includes symbionts and commensals that are found in or on marine animals, as well as many species that are pathogenic to animals (21). There are 12 species that are routinely isolated from human clinical samples, and the diseases in which they are implicated include diarrheal disease, septicemia, and wound infections (6).

Three species account for the majority of human Vibrio infections. Toxigenic Vibrio cholerae is the causative agent of the disease cholera and is acquired through ingestion of contaminated food or water. Infection may lead to a profuse, watery diarrhea that can lead to severe dehydration and death if left untreated. Worldwide, large outbreaks are caused by toxigenic strains of serogroups O1 and O139 that produce the cholera toxin, but in the United States, nontoxigenic strains predominate among the cases reported to the Centers for Disease Control and Prevention (CDC) (4). A total of 48 cases of cholera occurred in the United States in 2004 and were reported to the CDC (4), including eight cases infected with toxigenic V. cholerae, four of which were associated with travel, and 40 cases infected with nontoxigenic cholera. Vibrio cholerae accounted for 9.8% of all Vibrio isolates reported in the United States to the CDC in 2004 (4).

Vibrio parahaemolyticus is a halophilic bacterium that naturally inhabits coastal waters. Infection can result in acute gastroenteritis (6), and wound infections can occur as a result of exposing an open sore to warm seawater. Infection is rarely fatal, and the majority of cases arise from ingestion of undercooked seafood, especially raw oysters. A total of 240 cases (49.3% of Vibrio cases in the United States) were reported to the CDC in 2004 (4). Vibrio vulnificus is another pathogenic species that is naturally present in warm seawater. The typical route of infection is ingestion of raw oysters, but infection can occur through wounds that are exposed to seawater or infected organisms. The bacterium can cause wound infections, gastroenteritis, or septicemia (6). The species is an opportunistic pathogen that causes severe infections in individuals who are immunocompromised and in those with chronic liver disease; in about 50% of such cases fatal complications result from infection. A total of 92 isolates (18.9% of Vibrio isolates in the United States) were reported to the CDC in 2004 (4), of which 33% caused fatal infections. Although V. parahaemolyticus accounted for the majority of Vibrio infections in the United States, V. vulnificus was responsible for 82% of the fatalities in which a Vibrio species was implicated. Other Vibrio species that are routinely isolated from human clinical samples include V. alginolyticus, V. fluvialis, V. mimicus, Vibrio (Grimontia) hollisae, Vibrio (Photobacterium) damsela, V. furnissii, V. cincinnatiensis, V. harveyi, and V. metschnikovii. Of these species, V. mimicus was of special interest to us: it is closely related to V. cholerae, and so it can be difficult to differentiate the two species because they share many phenotypic characteristics (5). V. mimicus can also carry the cholera toxin gene as well as other virulence-associated genes that are used to identify V. cholerae (3).

Accurate phenotypic identification of Vibrio species is problematic, largely because of the great variability in biochemical characteristics (21). O'Hara et al. (16) evaluated six commercial systems for the ability to identify the 12 species of Vibrio found in clinical samples. They found that many isolates are not accurately identified by commercial methods, with the accuracy of systems ranging from 63.9% to 80.9% (16). Most members of the genus are halophilic, and the addition of NaCl is often required for enzymatic activity; however, the concentration of NaCl can affect the biochemical profile and lead to erroneous identification with at least one system (API 20E [13]). The characteristics shared by Aeromonas and Vibrio also result in classification of isolates in the wrong genus (1, 18). Additional drawbacks of biochemical methods are that they are time-extensive (i.e., reactions must incubate for an extended time period, often requiring 2 to 7 days to complete) and that the interpretation of results requires specialized training that may not be available to all laboratories.

Molecular methods that utilize the PCR and nucleotide sequence determination overcome many of the limitations of phenotypic methods. Molecular techniques, particularly nucleotide sequence determination, provide data that are objectively scored to provide an unambiguous identification. Nucleotide sequences can be placed into a phylogenetic framework, which allows an assessment of the genetic relationships among isolates and can reveal isolates that represent undescribed species. Most importantly, methods that utilize the PCR can lead to identification of an isolate within hours as opposed to days and can be used on small quantities of cells, including those that are not viable or are otherwise unculturable.

A rapid and accurate molecular method that identifies multiple species in one assay would be a useful tool for clinical laboratories and would enhance Vibrio surveillance and diagnosis. A simple molecular tool could also lead to better estimates of the incidence of Vibrio infections in the United States. While a total of 487 Vibrio isolates were reported to the CDC in 2004 (4), the incidence of Vibrio infections is likely to be much greater, particularly because only infections due to toxigenic V. cholerae serogroup O1 or O139 were nationally notifiable prior to 2006.

In this report, we present a two-step approach to identify Vibrio isolates that are pathogenic to humans: a multiplex PCR assay to identify the most commonly encountered Vibrio isolates (V. cholerae, V. parahaemolyticus, V. vulnificus, and V. mimicus) and a robust sequence-based approach using rpoB sequencing and phylogenetic analysis that can cluster isolates into other Vibrio species and can clearly differentiate Vibrio from other closely related genera, such as Aeromonas. The multiplex PCR primarily targets variation in conserved housekeeping genes. We also generated a Vibrio rpoB reference database and then determined nucleotide sequence for an 870-bp portion of the rpoB gene to identify Vibrio isolates that could not be classified on the basis of phenotype or the multiplex PCR. Sequence analysis of the rpoB gene has been proposed as a method for bacterial identification (15) and has been used to identify bacterial isolates in a number of taxa, including Pseudomonas (2), Corynebacterium (8), and the staphylococci (14). The combination of the multiplex PCR and rpoB sequencing allowed us to quickly and accurately identify the predominant Vibrio species that are pathogenic to humans, unambiguously place unidentified Vibrio isolates into species within a phylogenetic framework, and identify isolates that appear to represent as-yet-unnamed Vibrio species.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

 
A total of 316 bacterial isolates were used in the study: 309 Vibrio isolates, representing 26 named species, as well as isolates in the genera Photorhabdus (one), Salmonella (one), Yersinia (one), and Aeromonas (four). We included Vibrio (Grimontia) hollisae and Vibrio (Photobacterium) damsela among the 12 species that are pathogenic to humans. All isolates were obtained from the frozen culture collection in the Foodborne and Diarrheal Diseases Branch of the CDC. Isolates were stored at –70°C in tryptic soy broth with 20% glycerol.

PCR template preparation. Crude lysates were prepared for PCR as follows. Species pathogenic to humans were grown overnight at 35°C on tryptic soy agar plates with 5% sheep blood. Marine species were grown on marine agar plates and incubated at either room temperature or 20°C (V. logei) for 48 h. After incubation, a single colony was scraped from the plate's surface and suspended in 200 µl of 1x Tris-EDTA, pH 8.0. The suspension was then heated at 95°C for 10 min and centrifuged for 2 min to pellet cellular debris.

Development of the multiplex assay. We initially used information in GenBank (http://www.ncbi.nlm.nih.gov/GenBank/) to select target genes for the multiplex assay. Vibrio species were well represented for two variable genes in the database: hsp60 (11) and sodB (Yoshiyuki Yamada, unpublished data). Sequences for the two genes were downloaded from GenBank, and each was aligned with ClustalX (22). We visually searched for sequence regions that potentially could differentiate individual pathogenic species from other Vibrio spp., and candidate primer sites were identified for V. vulnificus, V. mimicus, and V. cholerae. Intraspecific variation within each site was then assessed for V. vulnificus in hsp60 (18 isolates; primers from reference 23) and for V. cholerae and V. mimicus in sodB (48 V. cholerae isolates and 19 V. mimicus isolates; primers courtesy of Yoshiyuki Yamada). For the V. parahaemolyticus marker, we downloaded four Vibrio genome sequences from GenBank (V. parahaemolyticus, NC 004603/NC 0046053; V. cholerae, NC 002505/NC 002506; V. fischeri, NC 006840/NC 006841; and V. vulnificus, NC 004459/004460) and then extracted and aligned sequences for the flagellin genes. Primers were designed to match the flaE sequence of V. parahaemolyticus. A highly conserved, positive internal control for the multiplex PCR was developed on the basis of an alignment of Vibrio and Enterobacteriaceae 16S rRNA gene sequences.

Optimization was performed for each primer pair individually and then all primers in combination. Final primer concentrations were adjusted to give approximately equal signals for each gene fragment. Each reaction contained 2 units of AmpliTaq Gold (Applied Biosystems, Foster City, CA), and final concentrations of each deoxynucleoside triphosphate (dNTP) and MgCl₂ of 0.2 mM and 1.5 mM, respectively, and primer concentrations ranged from 0.05 µM to 1 µM (Table 1). Thus, a typical 20-µl reaction mixture contained 1.5 µl crude lysate, 0.2 µl of AmpliTaq Gold, 2 µl dNTP stock (2 mM each), 2 µl 10x buffer, 1.2 µl MgCl₂ stock (25 mM), 2.9 µl double-distilled water, and the various amounts of each 10 µM stock of the primers: Vc primers, 1 µl each; Vm primers, 1.5 µl each; Vp primers, 2 µl each; Vv primers, 0.5 µl each; and 16S rRNA primers, 0.1 µl each. The thermal cycling profile was as follows: a 15-min soak at 93°C followed by 35 cycles of 92°C for 40 s, 57°C for 1 min, and 72°C for 1.5 min and a final soak at 72°C for 7 min. PCR amplicons were electrophoresed in 1.5% agarose for 1 h at 100 V and then visualized by ethidium bromide staining and UV illumination. The result for each isolate was determined by comparison to those for control isolates of V. mimicus, V. cholerae, V. vulnificus, and V. parahaemolyticus. Any isolate for which the original identification did not match our multiplex results, or which could not be classified based on the PCR or phenotypic characters, was subjected to rpoB sequence determination and phylogenetic analysis as described below.

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TABLE 1. Sequence of primers and final concentration and product size for each of five primer pairs in the multiplex PCR

rpoB amplification and sequence determination. We developed primers to amplify and sequence a 984-bp portion of the rpoB gene by first extracting rpoB sequences from Vibrio genome sequences and aligning them with Enterobacteriaceae sequences reported in the work of Mollet et al. (15). We used primers CM32b (15) and 1110F (5'-GTA GAA ATC TAC CGC ATG ATG-3') for amplification and two additional primers for sequencing (1661F, 5'-TTY ATG GAY CAR AAC AAC CC-3'; 1783R, 5'-GGA CCT TYA GGN GTT TCG AT-3'). The 50-µl PCR mixtures contained 2 units of AmpliTaq Gold, 1.5 µl of crude lysate, and the following reagents at the final concentrations listed in parentheses: primer (0.5 µM each), dNTPs (0.2 mM each), Betaine (1 mM) (Sigma-Aldrich, St. Louis, MO), and MgCl₂ (1.5 mM). The touchdown PCR was preceded by a 15-min soak at 93°C. The thermal cycle was as follows: 92°C for 40 s, 60°C to 50°C for 1 min (decreasing 1°C each cycle, then 50°C for 30 cycles), 72°C for 1.5 min, and a final soak at 72°C for 7 min. Agarose gel electrophoresis and ethidium bromide staining were used to check the quality and quantity of PCR product, and amplicons were then purified with the QIAquick PCR purification kit (QIAGEN, Valencia, CA). The product was quantified by comparison to a known quantity of the low-molecular-mass ladder (Invitrogen, Carlsbad, CA) on an ethidium bromide-stained gel. We used the DTCS Quickstart kit (Beckman Coulter, Fullerton, CA), following manufacturer's instructions with the following specifications: we used 1 µl of a 20 µM primer stock solution, 50 fmol of template, an annealing temperature of 50°C, and Betaine at a final concentration of 1 mM. Sequence determination was conducted on a Beckman CEQ 8000 Genetic Analysis system.

Chromatograms were imported into SEQMAN in the package DNAStar (Lasergene), assembled into contigs, edited, and exported as FASTA files. The rpoB sequences were aligned with ClustalX, and pairwise genetic distances were calculated using the Kimura two-parameter method in the program MEGA (10). The rpoB sequences from three published Vibrio genome sequences were also included in the analysis (7, 9, 12). A neighbor-joining tree was constructed from the genetic distance matrix, and the bootstrap confidence intervals for each node were calculated over 1,000 replicate trees.

Nucleotide sequence accession numbers. GenBank accession numbers for sequences generated in this study are EF 064367 to EF 064446.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

 
Multiplex results. The multiplex assay was tested on a collection of 316 bacterial isolates that included 309 Vibrio isolates. Among these 309 isolates were 190 isolates representing the four target species, isolates representing an additional 22 named species (see Table 2), and a total of 15 isolates that had been identified only to genus level. Representative gels are shown in Fig. 1. All 190 isolates of the four target species yielded the expected species-specific amplicon (Table 2). For three of the target species, the 16S rRNA amplicon was usually outcompeted by the species-specific amplicon, but both amplicons were usually visible for V. parahaemolyticus (Fig. 1). Only the 16S rRNA fragment was amplified from the isolates representing the remaining 22 species, as expected. Of the 15 isolates that were previously identified only to genus level, three (9564-98, 2410-00, and 2423-02) yielded the V. parahaemolyticus amplicon. Also, the V. parahaemolyticus amplicon was obtained for one isolate that was originally reported to the CDC as V. alginolyticus (F8891), a result that is not too surprising given the similar biochemical profiles of the two species (6). The classification of these four isolates as V. parahaemolyticus was confirmed using nucleotide sequencing and evolutionary analysis of a segment of the rpoB gene as described below. Thus, we found that the multiplex PCR rapidly and accurately discriminated four Vibrio species that account for the majority of Vibrio infections in the United States each year (4).

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TABLE 2. Summary of multiplex results for 316 bacterial isolates, listed by original species identification

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FIG. 1. Agarose gels showing results of the multiplex PCR. L, low-molecular-mass ladder. Controls are labeled as follows: A, no-DNA control; M, V. mimicus; C, V. cholerae; V, V. vulnificus; P, V. parahaemolyticus. Gel A: samples 1 to 3, V. mimicus; samples 4 to 10, V. cholerae serogroup O1 Ogawa, Inaba, non-O1, O139, O141, O22, and O155, respectively. Gel B: samples 1 to 6, V. vulnificus; samples 7 to 10, V. parahaemolyticus O4:K68, O3:K6, O3:K6, and O3:K48, respectively. Gel C: sample 1, V. alginolyticus; sample 2, V. cincinnatiensis; sample 3, P. damsela; sample 4, V. fluvialis; sample 5, V. furnissii; sample 6, V. harveyi; sample 7, V. hollisae; sample 8, V. metschnikovii; sample 9, V. splendidus; sample 10, V. campbellii. Note that the 16S rRNA control amplicon is usually outcompeted by the species-specific amplicons, but it is clearly visible in some samples, particularly samples of V. parahaemolyticus (e.g., gel A, V. cholerae sample 7, and gel B, V. parahaemolyticus samples 7, 9, and 10).

rpoB sequencing results. We obtained 870 bp of sequence from a total of 60 Vibrio isolates that had been classified by biochemical methods, 15 isolates that could not be classified on the basis of phenotype, and four isolates of other enteric bacteria that were included to root the phylogenetic tree. The rpoB gene was polymorphic within all 11 of the pathogenic Vibrio species for which we sequenced more than one isolate (Table 3). The synonymous substitution rate (d_S) was greater than the nonsynonymous substitution rate (d_N), as was expected for a conserved housekeeping gene (Table 3).

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TABLE 3. Genetic variation within 870 bp of the rpoB gene in 11 Vibrio speciesa

All Vibrio species included in the analysis were distinguishable from isolates of other species on the basis of their rpoB sequence (Fig. 2). The level of bootstrap support for each of the pathogenic species clusters was very high, ranging from 93% for V. fluvialis isolates to 100% for most of the other Vibrio species (Fig. 2). The closest species pair was V. harveyi and V. campbellii, which differed at two nucleotide sites (0.23%). Genetic distances for other closely related species pairs were 2.1% (V. fluvialis and V. furnissii), 3.2% (V. alginolyticus and V. parahaemolyticus), and 4.2% (V. cholerae and V. mimicus) (Table 4). The species clusters based on the rpoB phylogeny were consistent with the results based on sequencing of other loci (19).

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FIG. 2. Neighbor-joining tree constructed from Kimura two-parameter distances calculated from 870 bp of rpoB sequence. The numbers at each node indicate the percentage of bootstrap replications in which a particular node appears (shown for nodes where value was >50%). Isolates that were identified by rpoB sequence analysis are shown highlighted in gray and are designated by the original identification. The rpoB sequences for Escherichia coli K-12 and Shigella flexneri 2a were extracted from complete genome sequences downloaded from GenBank (accession numbers NC 000913 and NC 004337, respectively). GenBank accession numbers for sequences generated in this study are EF 064367 to EF 064446.

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TABLE 4. Average nucleotide distance between each pair of Vibrio species calculated for the rpoB genea

We sequenced a total of 15 isolates that were not classified by phenotypic methods because they differed in one or more biochemical characteristics from other Vibrio isolates. Information on the isolates is shown in Table 5. The sequences from 10 of the isolates fell within the core Vibrio group: four clustered with V. alginolyticus, three with V. parahaemolyticus, and three with the V. harveyi-related group. The sequences from the remaining five isolates were scattered in the tree, with one isolate clustering with V. anguillarum, one with V. fluvialis, and one with V. hollisae. Two of the Vibrio sequences did not cluster with any of the other groups in the tree. An isolate (F8891) that had been reported to the CDC as V. alginolyticus did not cluster with other sequences for that species but instead clustered with V. parahaemolyticus, which was consistent with the results from the multiplex PCR assay.

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TABLE 5. Characteristics of 15 isolates that were identified by rpoB sequence determination and phylogenetic analysis

The cluster of isolates including V. harveyi raised questions about classification of these closely related isolates. Thompson et al. (20) suggested that V. trachuri is synonymous with V. harveyi. The V. campbellii and V. harveyi isolates included here had rpoB sequences that differed by only two nucleotides. These two isolates differed from the cluster of "V. trachuri" and two unknowns by 15 to 19 nucleotides (1.8 to 2.2%), a level of divergence that is similar to that observed between V. fluvialis and V. furnissii and also between two distinct clusters of V. vulnificus (Fig. 2; Table 4). In this case, divergence alone cannot dictate a species boundary; however, based on the phylogeny shown here, principles of phylogenetic classification would require either that V. harveyi also include V. campbellii to avoid paraphyly in the species or that V. trachuri be a separate species along with V. campbellii and V. harveyi. Further study is needed to resolve the relationships between the isolates in the V. harveyi cluster.

The V. harveyi cluster also included isolate 9866-84, an isolate that was included because it could not be classified by phenotypic methods. The average genetic distance between 9866-84 and other isolates in the cluster was 4.2%, a value that was consistent with distances between closely related species (V. cholerae and V. mimicus, 4.2%; V. parahaemolyticus and V. alginolyticus, 3.2%). We suggest that this isolate is a species closely related to, but separate from, V. harveyi.

The rpoB sequences of two other clinical isolates (2419-00, from stool, and 2423-01, from blood) were divergent from all other Vibrio sequences included here (d > 13%) and did not cluster with any other sequences in the phylogenetic tree. Because our rpoB database does not yet include all described Vibrio species, we needed additional information to determine if these two isolates represented previously named Vibrio species. Thus, we sequenced a portion of the 16S rRNA gene and conducted BLAST searches of GenBank to assess the sequences' relatedness to other Vibrio isolates (data not shown). The two isolates did not closely match any named species on the basis of the 16S rRNA gene sequence: the closest named species to isolate 2419-00 was V. harveyi (980/986-bp match, 99% similarity) and to isolate 2423-01 was V. cholerae (1,515/1,545-bp match, 98% similarity). On the basis of the rpoB and 16S rRNA gene sequence analysis, we conclude that these two isolates likely represent unnamed Vibrio species.

The two-step approach that we have presented here allows rapid and accurate discrimination of the Vibrio species that are implicated in human infections. The advantages of our multiplex are that conserved housekeeping genes are used as a source of markers so that the targeted gene is present in all isolates of a species and multiple species can be discriminated with a single PCR assay. The assay is designed as a rapid classification tool for clinical laboratories, but the markers themselves could be incorporated into other PCR and DNA microarray assays that use virulence genes as markers for pathogenic species (e.g., reference 17). We note that this assay used alone would not assess the pathogenic potential of bacteria in environmental samples, where virulence determination is important in assessing the risk of human illness. We are currently developing molecular probes for the remaining Vibrio human pathogens and adapting the assay to a Luminex platform (Luminex Corporation, Austin, TX) to provide a single assay that can rapidly detect and identify the 12 species that are pathogenic to humans.

The rpoB gene also provides a useful identification tool; its advantages over the 16S rRNA gene are that it exists as a single-copy gene and it has sufficient phylogenetic signal to discriminate among all of the Vibrio species that we have sequenced to date. Thus, our rpoB reference database that we are assembling will augment the identification of Vibrio species by PCR assays and by sequence-based approaches under way in other laboratories (19).

 
* Corresponding author. Mailing address: Foodborne and Diarrheal Diseases Branch, Division of Bacterial and Mycotic Diseases, National Center for Infectious Diseases, Centers for Disease Control and Prevention, 1600 Clifton Road NE, Mailstop C-03, Atlanta, GA 30333. Phone: (404) 639-2011. Fax: (404) 639-3333. E-mail: crt6{at}cdc.gov.

Published ahead of print on 8 November 2006.

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Journal of Clinical Microbiology, January 2007, p. 134-140, Vol. 45, No. 1
0095-1137/07/$08.00+0     doi:10.1128/JCM.01544-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

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